The controlled expression of two or more proteins at a defined and stable ratio remains a substantial challenge, particularly in the bi- and multispecific antibody field. Achieving an optimal ratio of protein subunits can facilitate the assembly of multimeric proteins with high efficiency and minimize the production of by-products. In this study, we propose a solution based on alternative splicing, enabling the expression of a tunable and predefined ratio of two distinct polypeptide chains from the same pre-mRNA under the control of a single promoter. The pre-mRNA used in this study contains two open reading frames situated on separate exons. The first exon is flanked by two copies of the chicken troponin intron 4 (cTNT-I4) and is susceptible to excision from the pre-mRNA by means of alternative splicing. This specific design enables the modulation of the splice ratio by adjusting the strength of the splice acceptor. To illustrate this approach, we developed constructs expressing varying ratios of GFP and dsRED and extended their application to multimeric proteins such as monoclonal antibodies, achieving industrially relevant expression levels (>1 g/L) in a 14-day fed-batch process. The stability of the splice ratio was confirmed by droplet digital PCR in a stable pool cultivated over a 28-day period, while product quality was assessed via intact mass analysis, demonstrating absence of product-related impurities resulting from undesired splice events. Furthermore, we showcased the versatility of the construct by expressing two subunits of a bispecific antibody of the BEAT (R) type, which contains three distinct subunits in total.

We describe a mammalian expression construct (SPLICELECT™) that allows the redirection of a proportion of a secreted protein onto the cell surface using alternative splicing: whereas the majority of the RNA is spliced into a transcript encoding a secreted protein, a weak splice donor site yields a secondary transcript encoding, in addition, a C-terminal transmembrane domain. The different sequence elements can be modified in order to modulate the level of cell surface display and of secretion in an independent manner. In this work, we demonstrated that the cell surface display of stable cell lines is correlated with the level of the secreted protein of interest, but also with the level of heterodimerization in the case of a bispecific antibody. It was also shown that this construct may be useful for rapid screening of multiple antibody candidates in binding assays following transient transfection. Thus, the correlation of product quantity and quality of the secreted and of membrane-displayed product in combination with the flexibility of the construct with regards to cell surface display/secretion levels make SPLICELECT™ a valuable tool with many potential applications, not limited to industrial cell line development or antibody engineering.

Cell populations that are exclusively composed by descendants from a defined single parental progenitor are referred to as "monoclonal", "clonal" or more correctly as "clonally derived cell population". Clonal derivation of cell lines used in the manufacturing of recombinant biologics is a regulatory requirement that aims to ensure a robust process and consistent quality throughout the life cycle of a product. Clonal derivation of cell lines is usually ensured by the process (e.g. two subsequent rounds of limiting dilution). Here we present an approach to analytically assess the probability of clonal derivation of existing cell populations. Using target locus amplification (TLA) followed by next generation sequencing (NGS), unique genetic features can be identified, for example the integration site of the plasmid in the host cell genome or plasmid-plasmid junctions of the plasmids used for expression of recombinant biologics. Whereas a direct assessment of clonal derivation using TLA/NGS data is challenging due to limitations in specificity, confirmed clonally derived populations generated from the cell line population can be analyzed by qPCR for the presence of the unique genetic features identified by TLA/NGS. In the present study, a statistical analysis allowed the demonstration that two independently generated CHO cell lines were clonally derived with an upper 95% confidence interval limit of a potentially present contaminating population of 1.3%.

Background Naturally occurring chromatin modifying elements such as MAR, UCOE or cHS4 were identified from the genome of higher eukaryotes. It is well documented that the presence of these regulatory elements leads to better recruitment of transcriptional machinery and/or prevents epigenetic silencing mechanisms [1,2]. The goal of this work was the evaluation of a new genetic element to improve transgene expression, from the flanking sequences of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, which is a ubiquitously expressed enzyme. It was hypothesized that the surrounding genetic environment of the gene may lead to a DNA structure favorable for transgene expression in eukaryotes. In this study, we have investigated the effect on stable and transient expression in CHO and HEK293 cells of the 3.2 kilo base pairs (kb) sequences flanking upstream (5’) and downstream (3’) the GAPDH locus.

Cette invention concerne une construction d'expression pour l'expression de polypeptides chez des cellules hotes faisant appel a l'epissage alternatif. Cette construction d'expression peut etre utilisee pour l'expression de polypeptides tels que des anticorps, des fragments d'anticorps et des anticorps bispecifiques par expression des produits geniques requis pour exprimer la proteine dans une proportion conduisant aux titres les plus eleves ou au meilleur profil de qualite de produit.

BACKGROUND:CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies.METHODS:GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401.RESULTS:GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization.CONCLUSION:These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Background The binding of two biological targets with a single IgGbased molecule is thought to be beneficial for clinical efficacy. However the technological challenges for the development of a bispecific platform are numerous. While correct pairing of heterologous heavy and light chains (Hc and Lc) can be achieved by engineering native IgG scaffolds, crucial properties such as thermostability, effector function and low immunogenicity should be maintained [1]. The molecule has to be expressed at industrially relevant levels with a minimum fraction of contaminants and a scalable purification approach is needed to isolate the product from potentially complex mixtures. This article introduces a novel bispecific platform based on the proprietary BEAT technology (Bispecific Engagement by Antibodies based on the T cell receptor) developed by Glenmark.

The screening of high-producing cells is a time consuming procedure. Starting from a single cell after a single cell dilution the procedure takes weeks to complete. The affinity matrix method is a technique for screening a cell population for high producers. Using this method it is possible to perform bulk sorting via FACS and get a complete sub population containing high producing cells instead of single cells. The affinity matrix is a "molecular anchor" which captures the secreted product via a specific antibody immobilized on the cell surface. In this manner the captured product can be detected with a fluorescence labeled antibody. Cells showing a high fluorescence after this procedure have numerous products bond on their surface and are supposed to be high producers. The present work describes a variant of the affinity matrix method which can be used for antibody secreting cells. The antibodies become captured on the cell surface by immobilized protein A instead of high priced capture antibodies. We show that these affinity matrix construct is functional on the cell surface of hybridoma cells. Also we prove the bonds of the components via protein micro array and can confirm that no unspecific binding within the construct occurs.

The increasing demand for human mesenchymal stem cells (MSCs) for cell-based therapies and tissue engineering leads to tremendous research effort to find novel sources of MSCs. Due to its easy availability and high frequency of MSCs, the human umbilical cord (UC) presents a promising and non-controversial source for these cells. We developed protocols for the xeno-free isolation and expansion of UC-MSCs. In this study, the stability of the isolated cells during ex-vivo expansion and the response to osteogenic stimulation was investigated using flow cytometry. Our results indicate that the isolated UC-MSCs can be expanded with a stable immunophenotype at least for 28 days under our cultivation conditions. Interestingly, we observed a decrease of the expression of the surface antigen Thy 1 (CD90), when the cells were induced to differentiate into the osteogenic lineage. These findings suggest that the CD90 down regulation is an early osteogenic feature.

Embryonic Stem (ES) cells are self-renewing and pluripotent cells that can differentiate into a variety of cell lineages. Implementation in tissue engineering or as model systems for drug discovery makes ES cells attractive as a cell source. To use ES cells for these applications, technologies are required to generate a large number of cells with defined characteristics. Currently, adherent culture methods are routinely applied for the maintenance of undifferentiated ES cells. However, perfused and stirred bioreactors enable a more homogenous environment and, more importantly, facilitate the ability to monitor and control culture parameters (e.g. oxygen content and pH) which is advantageous compared with static culture vessels. In this work, four different suspension cultivation systems (two spinner flasks with different stirrer design, an Erlenmeyer flask and a Petri dish) were compared with respect to the ability to generate undifferentiated ES cells. Therefore, the murine ES cell line E14.1, 129/Ola with an eGFP transgene targeted to the Brachyury locus was used. Besides the evaluation of biomass production, pluripotency marker expression was analyzed applying flow cytometry.

Notch signalling regulates arterial differentiation during embryonic development. The Notch ligand Delta-like 1 (Dll1) is known to be an essential regulator of postnatal arteriosclerosis (Limbourg et al. 2007). Clarification of its role within cardiac repair may lead to new ways of improving cardiovascular regeneration through targeted manipulation of cardiovascular differentiation. Since the soluble form leads to inactivation (Nichols et al. 2007), a fusion protein of the extracellular domain of the Notch ligand Delta-like 1 and the Fc region of IgG has been selected to be recombinantly produced in mammalian cells. CHOSFS cells were used as the expression system in this work. They were chemically transfected and cultured in serum-free medium under selective conditions. High-producing clones were gained by single-cell cloning via FACS and via dilution method. For FPLC-purification protein G as well as protein A columns were used. The activity was verified using a luciferase assay utilizing human umbilical cord vein endothelial cells (HUVEC).

Up to 2.8 × 10 7 fibroblast-like cells displaying an abundant presence of mesenchymal stem cell (MSC) markers CD73, CD90, CD105 and a low level of HLA-I expression can be isolated from one whole human umbilical cord (UC) using a simple and highly reproducible explant culture approach. Cells derived from whole UC, similar to cells collected from separate compartments of UC, display a distinct chondrogenic and adipogenic potential. Therefore they are potential candidates for cartilage and adipose tissue engineering. Cell differentiation along the osteogenic pathway is, however, less efficient, even after the addition of 1.25-dihydroxyvitamin D3, a potent osteoinductive substance. Isolated cells are highly proliferative, tolerate cryopreservation with an average survival rate of about 75% and after thawing can be propagated further, at least over 20 population doublings before their proliferative activity begins to decline. More importantly, they synthesize numerous trophic factors including neurotrophins and factors which facilitate angiogenesis and hematopoiesis. In conclusion, cells isolated from whole UC satisfies all requirements essential for the generation of stem cell banks containing permanently available cell material for applications in the field of regenerative medicine. Nevertheless, further studies are needed to improve and adjust the methods which are already employed for adult MSC expansion and differentiation to specific properties and requirements of the primitive stem cells collected from UC. So, our data verify that the choice of individual parameters for cell propagation, such as duration of cell expansion and cell seeding density, has a substantial impact on the quality of UC-derived cell populations.

Galway, Ireland Investigation of the Osteogenic Potential of Mesenchymal Stromal Cells derived from Human Umbilical Cord Tissue Pierre Moretti, Stefanie Boehm, Antonina Lavrentieva, Tim Hatlapatka, Ralf Hass, Thomas Scheper, Cornelia Kasper Corresponding Author: Kasper @iftc.uni-hannover.de 1 Institute of Technical Chemistry, University of Hannover, Hannover, Germany 2 Laboratory of Biochemistry and Tumor Biology, Dept. of Obstetrics and Gynecology, Medical University, Hannover, Germany

Galway, Ireland Subpopulations in Mesenchymal Stem Cell-Like Cultures from Human Umbilical Cord: Identification and Characterization Tim Hatlapatka, Ingrida Majore, Pierre Moretti, Ralf Hass, Cornelia Kasper Corresponding Author: kasper@iftc.uni-hannover.de Leibniz University of Hannover, Institute of Technical Chemistry, Callinstraße 5, D-30167 Hannover Hannover Medical School, AG Biochemie und Tumorbiologie, Klinik für Frauenheilkunde und Geburtshilfe, Carl-Neuberg-Str. 1, D-30625 Hannover

The improvement of specific productivity is a continuous challenge for bioprocesses involving mammalian cells, and hence, high-throughput methods and low-cost strategies are needed for the selection of high producers. The aim of this study was the productivity improvement of the hybridoma cell line IV F 19.23. For this purpose, a cell surface affinity matrix assay was established to identify and select high producers. This assay is based on the binding of secreted monoclonal antibodies to an affinity matrix assembled on the outer cell membrane. A protein microarray approach was used to investigate and optimize the functionality of the affinity matrix. The protein microarray was particularly useful to identify critical steps of the staining method, such as unspecific binding, before it was applied to the hybridoma cell line. Secreting hybridomas were treated with the affinity matrix and then selected via flow-cytometric cell sorting in four consecutive bulk sort rounds. The applied bulk strategy, allowing low screening costs, resulted in a 125% increase in specific productivity of the cell line in comparison to the initial population.

First isolated from bone marrow, mesenchymal stem or stromal cells (MSC) were shown to be present in several postnatal and extraembryonic tissues as well as in a large variety of fetal tissues (e.g., fatty tissue, dental pulp, placenta, umbilical cord blood, and tissue). In this study, an optimized protocol for the expansion of MSC-like cells from whole umbilical cord tissue under xeno-free culture conditions is proposed. Different fetal calf sera and human serum (HS) were compared with regard to cell proliferation and MSC marker stability in long-term expansion experiments, and HS was shown to support optimal growth conditions. Additionally, the optimal concentration of HS during the cultivation was determined. With regard to cell proliferative potential, apoptosis, colony-forming unit fibroblast frequency, and cell senescence, our findings suggest that an efficient expansion of the cells is carried out best in media supplemented with 10% HS. Under our given xeno-free culture conditions, MSC-like cells were found to display in vitro immunoprivileged and immunomodulatory properties, which were assessed by co-culture and transwell culture experiments with carboxyfluorescein diacetate succinimidyl ester-labeled peripheral blood mononuclear cells. These findings may be of great value for the establishment of biotechnological protocols for the delivery of sufficient cell numbers of high quality for regenerative medicine purposes.